Summary: The research of the Hematopoiesis Section is focused on the basic biology of stem cells and the use of stem cells as vehicles for cell and gene therapy. Hematopoietic stem cells (HSC) are a rare population of self-renewing cells that give rise to all cells in the peripheral blood, making them ideal vehicles for gene replacement therapy of inherited hematopoietic diseases. Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We have shown that Hmgb3 is a protein the binds both to transcription factors and to chromatin. We have used an Hmgb3 knockout mouse model to demonstrate that Hmgb3 is required to regulate the balance between differentiation and proliferation of the most primitive hematopoietic cells. We have shown that Hmgb3 regulates the expression of c-kit receptor on active HSC. We have made a marked Hmgb3 gene and are using Chromatin Immune precipitation (ChIP) to investigate the binding of Hmgb3 at the c-kit locus and to identify other DNA targets of Hmgb3. and direct immune precipitation to identify protein partners of Hmgb3. Specific Aim 1.2: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. We have identified a gene - Asridj, which is expressed in both skeletal muscle stem cells, Es cells and HSC, as well as being in several ?stem cell? databases. We are initiating experiments in which lentiviral transfer of the Asridj gene into bone marrow HSC will be used to identify the effects of over expression of Asridj. BACs have been isolated for conditional knockout experiments. Project 2: Gene therapy for the hemoglobinopathies. Gene transfer to HSC has recently been shown to cure Severe Combined Immune Deficiency, demonstrating that HSC gene therapy could be applied to more common diseases. We would like to develop a gene therapy for Sickle Cell Disease. However, current levels of gene transfer to HSC are too low to treat this disease. We have found that one important reason that gene transfer is so low is that the conventional retrovirus receptors on HSC are nearly undetectable. A second problem has been the instability of retrovirus vectors containing globin genes. Specific aim 2.1: We have shown that the receptors of the RD114 and FeLV-C retrovirus are expressed at high levels on hematopoietic stem cells, and that this leads to improved gene transfer to human hematopoietic cells in the sheep xenograft model. We are mapping the insertion sites of the FeLV-C and RD114 pseudotyped viruses to determine if these sites are as safe as those produced by conventional viral vectors. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells can be generated that will allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia. Our evaluation of the relative level of expression of red cell gene promoters using a transgenic mouse assay has shown that the AE-1 promoter linked to a chicken insulator element directs position independent, uniform, high-level, and copy number dependent expression. We have also identified a key regulatory region in the ankyrin promoter that we will modify in an attempt to increase the activity of the ankyrin promoter to give higher levels of globin expression. We have also demonstrated a compact insulator element in the ankyrin promoter that provides protection from gene silencing in vitro and in vivo. We are currently evaluating the regulatory regions of other red cells genes to identify both enhancers and barrier elements to incorporate into our globin vectors.